The goal of this project is to define the mechanism that determines Xist mRNA stability. Based on previous work by the investigator, the structure of the murine Xist gene has been revised to include new information on the 3' end of the gene. The investigator has shown that this area of the gene is significantly larger than previously reported. Sequence comparison between mouse and human revealed sequence similarity in the new 3' ends. Using both Northern analysis and RNAse protection experiments, the investigator has confirmed that both Xist mRNA isoforms are produced by a mechanism involving differential polyadenylation. These polyadenylation sites are located in the new 3' sequences identified. Additional expression studies have shown that Xist mRNA isoforms are developmentally regulated, and therefore show different stabilities. This pattern of expression suggests that regulatory elements may interact differentially with each mRNA isoform, which in turn may influences Xist expression early in development. The data suggest that Xist mRNA isoforms change independently of Tsix and that the developmental regulation of the Xist isoforms is influenced by mRNA stabilization during the period in which upregulation of the chosen X chromosome is observed. The specific mechanism responsible for Xist stability remains poorly characterized. Although early in development Xist is unstable, after development Xist is exceptionally stable (T1/2 = >5 hr). Recent data have placed in doubt the importance of the 5' end of Xist in this regulation. The investigator plans to explore the role of the new 3' end of Xist in gene stability. He also proposes to pursue the mechanism of developmental regulation of Xist stability from two directions. First, a complete a mutational analysis of Xist and the regions immediately adjacent to Xist will be done. Second, the investigator plans to evaluate genes in the methylation pathway and define their role in determining Xist mRNA stability.